Summary/Abstract Several neurodegenerative diseases are characterized by the unintended accumulation of toxic proteins, and some transformed cells induce the degradation of tumor suppressor proteins. Understanding the mechanisms that undermine proteasomal degradation in disease will help design targeted approaches to interfere in the pathological processes. The concentrations of cellular proteins are determined by the balance of synthesis and degradation, and most of the regulated degradation occurs by the Ubiquitin Proteasome System (UPS). The long-term goal of this research project is to develop a quantitative understanding of the UPS. The protease at the center of the UPS, the proteasome, degrades regulatory proteins and removes misfolded and damaged proteins as part of the cell's stress response. Thus, the proteasome faces the challenge of having to be able to degrade any protein but doing so with exquisite specificity. Proteins are targeted to the proteasome by ubiquitin tags but ubiquitin is also used as a signal in many other cellular processes. We therefore do not fully understand exactly how the tags specify the fate of a protein. We propose to elucidate the missing part of the protein degradation code. The degradation signal, or degron, contains a second component in the form of a disordered region in the target protein at which the proteasome initiates degradation. The proteasome has pronounced preferences for the amino acid sequence of the initiation site and we propose that recognition of the initiation region represents the second component of the degradation code. We will characterize this initiation code and investigate how it interacts with the ubiquitin code to determine the fate of proteins. In Aim 1 we will define the rules by which the proteasome selects its substrates at the initiation step of degradation. We will employ a large screen of a library of all human protein coding sequences to determine the proteasome's amino acid sequence preferences at initiation. We will also test how the sequence preferences affect the degradation natural proteins in cells, and investigate how the protein that accumulates in Huntington's Disease escapes proteasomal degradation. Aim 2 investigates how the proteasome recognizes ubiquitin tags during degradation and how this recognition step and the initiation step affect each other to tune protein degradation rates. We will also investigate how the relationships we discover affect natural proteins and processes in cells. The results of these experiments can ideally be used to design therapies for neurodegenerative diseases and other conditions caused by defects in protein degradation.